Silencing of integrin α5β1 promotes FCL and RA formation. (A) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for active integrin β1 (12G10 antibody). Representative TIRF images. (B) Analysis of 12G10 fluorescent intensity. N = 7 from one representative experiment. Similar results were observed from two individual experiments. One-way ANOVA with Tukey’s multiple comparison F(2, 13) = 149.9, P < 0.0001. (C) Western blots showing integrin β1 silencing efficiency and the effect on integrin β5 protein levels. U2Os cells silenced for integrin β1 shRNAs (shITGB1 #1, #2) or control shRNA were blotted for integrin β1, integrin α5, and α-tubulin. Representative blots out of two individual experiments. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for integrin α5 with two different shRNAs (shITGA5 #1, #2) or control shRNA were plated on FN-coated dishes and stained for integrin α5 (SNAKA51 antibody). Representative TIRF images. (E) Analysis of SNAKA51 fluorescent intensity from widefield microscopic images. N (images): shScr = 24, shITGA5 #1 = 20, shITGA5 #2 = 19 from one representative experiment. Similar results were observed in two individual experiments. One-way ANOVA with Tukey’s multiple comparison, F(2, 60) = 1, P < 0.0001. Data are the mean ± SD, *** P < 0.001. Scale bars, 10 µm. Source data are available for this figure: SourceData FS4.